Enzyme I: GP, AP[unreadable] [unreadable] Deuterium labeling of proteins is a commonly used technique in structural studies. We would like to explore how deuterium labeling affects the stability and activity of enzymes. As a model protein in ths study we have selected the first enzyme of an important new nitrogen signal pathway of E. Coli, the enzyme I-Ntr (EI-Ntr). The His356 residue of EI-Ntr is phosphorylated in the autocatalytic reaction of EI-Ntr with PEP/Mg(II) and this high energy phosphate is transferred to His16 of NPr, a small carrier protein containing 90 amino acids. We have obtained thermodynamic parameters for dimerization of EI-Ntr and for interactions of EI-Ntr and its monomeric amino terminal domain EIN-Ntr with the acceptor protein NPr using isothermal titration calorimetry, analytical ultracentrifugation and light scattering methods. Comparing the stability of EI-Ntr with its deuterium labeled counterpart we observed that the stability of deuterated enzyme is compromised and the Tm of the deuterated EI-Ntr is lowered by 4 degrees K as shown by the differential scanning calorimetry. We continue to thermodynamically characterize interactions of the deuterated EI-Ntr with its ligands to understand what aspects of the activity of this enzyme are affected by deuterium labeling.[unreadable] [unreadable] [unreadable] Oligomeric state of the equine infectious anemia virus (EIAV) matrix protein. (GP, NT)[unreadable] [unreadable] Equine infectious anemia virus (EIAV) matrix protein domain (MA) is targeting the viral precursor polypeptide to the cytosolic side of the infected cell membrane during the virus maturation. Despite poor sequence homology, MA domains from various retroviruses are similar in size (14-15 kDa) and share a highly conserved tertiary structure composed of five helices. Interestingly, HIV-1 MA crystallized as a trimer while EIAV MA was crystallized in a nonsymmetric dimeric unit. Nevertheless, fluorescence studies suggest that EIAV MA exists as a multimer of two to three subunits. We carried out a series of analytical ultracentrifugation measurements to characterize the oligomeric form of EIAV MA in solution. Our results are consistent with a primary monomer-trimer equilibrium model for EIAV MA at micromolar concentrations.[unreadable] [unreadable] [unreadable] Conformational dynamics of the ClpA oligomers. (GP, MRM)[unreadable] [unreadable] ClpA, a Hsp100/Clp chaperone from E. Coli, forms a hexameric ring of subunits that unfolds native proteins in an ATP-dependent process and delivers the unfolded polypeptides to ClpP protease for degradation. The precise mechanism of ATP driven unfoldase activity of ClpA is important but not well elucidated. Previous calorimetric titrations of ClpA hexamer with SsrA target peptide have suggested that peptide binding may trigger conformational changes within the ClpA hexamer. This observations were confirmed by analytical ultracentrifugation studies indicating that ClpA hexamer conformational changes induced by SsrA peptide binding are reflected by sedimentation coefficient distributions.[unreadable] [unreadable] We continue to use this methodology to gain further insight into the role of individual ClpA domains. Each subunit of the ClpA hexamer has two ATP binding domains (D1 and D2) and the N domain located at the end of the central channel. Using the ClpA-deltaN and the ClpA-ND1 mutants, lacking the N-terminal domain and the ClpP docking D2 domain, respectively, we were able to establish that the ring-forming D2 domain, rather then the presumably flexible N domain, is responsible for most of the conformational changes associated with substrate binding by the ClpA hexamer. Models of the ClpA substrate processing mechanism based on those results were further supported by the data of the ATP-ase activity stimulation obtained with ClpA deletion mutants. In those experiments proteins showing large conformational changes by analytical ultracentrifugation were also stimulated in their ATP-ase activity and vice versa.[unreadable] [unreadable] [unreadable] Capping Protein - CARMIL Interaction Studies. (GP, TU, JAH)[unreadable] [unreadable] Capping Protein (CP) is a highly conserved actin-binding protein that is essential for normal actin dynamics which is an important part of many cellular processes, including immunological responses and cancer cell proliferation. CP binds to the barbed end of the actin filament blocking both association and dissociation of actin monomers. One potential regulator of CP is CARMIL, that might act as potent CP antagonist and inhibit CP interaction with actin filaments.[unreadable] [unreadable] Two regions of the CARMIL protein (CAH3-a and CAH3-b) were previously identified as crucial for interactions with CP. Cloned fragments of CARMIL with sequences encompassing both the CAH3-a and CAH3-b regions, with several single point mutations with either of them, were used to study CARMIL interaction with mouse Capping Protein (mCP) by Isothermal Titration Calorimetry (ITC). Results obtained by ITC were compared with those from an actin polymerization inhibition assay allowing for the identification of CARMIL regions contributing to binding affinity and those necessary for uncapping activity. We continue to use this effective strategy to locate mCP active sites for CARMIL binding by studying several single point mCP mutants in ITC titrations. Results of those experiments are compared with fluorescence titrations where the intrinsic Tryptophane fluorescence of mCP is used.